Lymphatic vascular cell traffic from the periphery to lymph nodes occurs during both inflammatory responses as well as neoplasia. In both cases, unique chemokines (chemotactic cytokines) produced by the lymphatic endothelium play central roles in driving the respective transit of either immune cells or tumor cells. This proposal examines the genetic importance of glycosaminoglycans, an important class of complex carbohydrates that includes heparan sulfate (HS) and chondroitin sulfate (CS), in mediating chemokine- dependent cell traffic in the lymphatic microenvironment. These sulfated glycan chains are tethered to unique cell-surface bound as well as secreted proteoglycan core proteins, and they interact with basic amino acid-rich domains of several chemokines. The specific focus herein is on glycosaminoglycans produced by the lymphatic endothelium, as a preliminary body of work suggests that lymphatic HS (and possibly CS) mediate two critical chemokine functions: (i) the scaffolding of major lymphatic chemokines (such as CCL21 and CXCL12) in peri-lymphatic spatial gradients that appear to be held by the glycans, and (2) the unique presentation of chemokines to their cognate receptors on trafficking cells, wherein secreted lymphatic glycosaminoglycans may serve as chemokine co-receptors. This work will focus on the setting of neoplasia, where lymphatic chemokine-dependent trafficking involves both carcinoma cells as well as immune cells (with focus herein on dendritic cell migration). This proposal addresses the hypothesis that genetic disruption of lymphatic glycosaminoglycan biosynthesis will alter the ability of tumor as well as dendritic cells to migrate toward lymphatic vasculature and traffic to lymph nodes in a chemokine-dependent manner. The goals are to: (1) Identify proteoglycan core proteins produced by the lymphatic endothelium, and characterize the roles of glycosaminoglycan chains in establishing lymphatic chemokine gradients. Upon identifying the repertoire of proteoglycan core proteins that tether lymphatic endothelial HS and CS chains, the glycan chains will be purified and tested for their ability to bind to major lymphatic endothelial chemokines. In lymphatic endothelial cell- and matrix-based assays, the effects of genetically altering glycosaminoglycan biosynthesis on chemokine gradient formation in static as well as flow conditions will be examined. (2) Assess the genetic importance of lymphatic glycosaminoglycans in chemokine-dependent migration of tumor and dendritic cells. Lymphatic glycosaminoglycan biosynthesis will be targeted in murine Cre-LoxP models and in siRNA-altered human lymphatic endothelium, and the ability of tumor and dendritic cells to migrate toward the targeted endothelium will be determined. In addition, the ability of conditioned medium harvested from the mutant cells to oligomerize (cluster) lymphatic chemokines as well as support chemokine-mediated activation of tumor and dendritic-cell migration signaling pathways will be examined. (3) Characterize real-time cell trafficking in the setting of genetic alterations that target glycosaminoglycan fine structure in the lymphatic microenvironment in vivo. Trafficking of tumor and dendritic cells from peripheral lymphatic vessels to regional lymph nodes will be examined in mice bearing gene defects in lymphatic glycosaminoglycan biosynthesis. In separate carcinoma models in the gene-targeted mice, lymph node colonization by trafficking tumor and dendritic cells will also be examined. Additional mechanistic studies will examine chemokine distribution and receptor binding in vivo. This work may uncover a novel mechanistic paradigm for how the distribution and actions of multiple chemokines may be controlled by glycans in the lymphatic microenvironment. It may also establish a rational basis for novel therapeutic drug discovery. PUBLIC HEALTH RELEVANCE: Chemokines are "sensing" molecules that drive migration of specific cells toward a chemokine source. In states of disease such as inflammation or cancer that commonly afflict the Veteran population, chemokines produced by blood or lymph vessels may drive the transit of inflammatory or tumor cells with deleterious consequences. In the case of lung cancer, a leading cause of Veteran suffering and death, the consequences may include the spread of cancer to lymph nodes as well as chemokine-driven movement of specialized ("dendritic") immune cells to the nodes, which in turn can promote immune "tolerance" to the tumor. Preliminary work shows that a class of complex sugar molecules (glycosaminoglycans) may underlie or mediate the actions of such lymphatic chemokines. Herein, we block the production of such complex sugars in lymphatic vessel cells using state-of- the-art genetic tools, and examine the effects on dendritic cell migration and lymph node metastasis. This work may lead to novel therapy to inhibit the spread, morbidity, and mortality of cancer in our Veteran population.